The airway epithelium plays a crucial role in protecting the underlying connective tissue (CT) from noxious agents. Damage and shedding of the epithelium are observed in the airways of asthma, cystic fibrosis and rhinitis patients. The aim of this study was to investigate the mechanisms by which epithelial damage occurs, and the consequences of such damage for the inflammatory process in the airways. In this study, cultured normal human bronchial epithelial cells, excised rat tracheae, and cultured murine mast cells were used as model systems. Metabolic alterations, morphological changes and cell-cell contact stabilities were investigated. The T-helper (Th)-1 cytokines, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin (IL)-1β were found to be pro-inflammatory, leading to major morphological changes, inhibitions in desmosome formation, and accelerated cell death. The Th2 cytokines, IL-4, IL-5, and IL-13 were found to cause no changes in cell death, nitric oxide levels and desmosome formation but instead an increase in proliferation, therefore were anti-inflammatory in this respect. Increasing the osmolarity of the airway surface liquid (ASL) altered the integrity of the tight junction (TJ) and allowed a 4-kDa compound to penetrate the epithelial layer and access the CT. This effect was reversible if the ASL was returned to 295 mOsm. Intentionally breaking the TJ with EGTA and subsequent osmolar changes in ASL demonstrated the importance of TJ and the fragility of the CT under hyperosmolar stress, leading to a disrupted CT with larger capillaries and altered elemental ion content and epithelial denudation. Hydrocortisone was shown to downregulate IL-4-induced IL-6 upregulation in murine mast cells. Furthermore, incubating mast cells with hydrocortisone lead to a new subpopulation that was morphologically unique, that displayed new cell surface markers (CD44 and CD61) and that lacked CD54. These changes modify the interactions of mast cells with surrounding cells in the CT and epithelium. In conclusion, the balance between pro- and anti-inflammatory cytokines and ASL osmolarity may influence the role of the airway epithelium as a barrier. The pharmacological use of hyperosmolarity to disrupt TJ reversibly may help facilitate the delivery of drugs through the airway epithelium.

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

The vagina is the port of entry for sexually transmitted diseases in women. Its epithelium constitutes the luminal border, thus comprising an important defence barrier. The objective of this work was to investigate the mechanisms of importance in the immune defence of the vaginal epithelium of healthy, fertile women, and possible menstrual cycle changes. Effects of hormonal contraceptive usage on oestrogen receptor (ER) and progesterone receptor (PR) expression were studied. The contribution of epithelial cell to the immune defence was estimated by assaying their expression of antimicrobial defensins and the epithelial thickness. Vaginal biopsies and serum samples were collected during the follicular and luteal phases in regularly menstruating women (controls) and in users of combined oral contraceptives (COCs), levonorgestrel implants (LNGs), or depot-medroxyprogesterone acetate injections (DMPAs). Fifteen healthy women (aged 20–34 years) were enrolled in each group. Morphometry was performed on vaginal tissue stained with haematoxylin/eosin and by immunohistochemistry using monoclonal antibodies against immune cell markers, PR, and ER. Expression of mRNA for human α-defensins HD-5 and HD-6, and human β-defensins (HBD) 1 to 4 were determined by real-time qRT-PCR and in situ hybridization. In controls, the epithelium was 261 ± 16 μm thick and harboured 241 ± 35 leukocytes (CD45+) per mm2. T lymphocytes (CD3+) dominated. Both αβ T cells and γδ T cells were present with an approximate 4-fold dominance of αβ T cells. Cytotoxic T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7 ± 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes and granulocytes were generally absent. No differences were found between the follicular and luteal phase. All four β-defensins analysed for were detected in vaginal epithelium and most samples expressed at least two. HBD-2 and HBD-3 were most frequent. HBD-3 and HBD-4 expressing cells were localized in the parabasal and intermediate cell layers. α-defensins were not detected. The epithelium was significantly thicker (333 ± 9 μm) in COC, LNG, and DMPA users than in controls, and commonly showed hyperplasia. In DMPA and LNG users the frequency of intraepithelial leukocytes (CD45+) was increased, explained by increased frequencies of both αβ and γδ T cells. In DMPA users there was also a selective increase in CD8+ T cells. PR expression was significantly reduced in DMPA users compared with controls, COC and LNG users. COC and particularly DMPA users often had undetectable levels of serum E2. In conclusion, both adaptive immunity, i.e. intraepithelial T cells, and innate defence mechanisms, i.e. intraepithelial macrophages and β-defensins, are believed to contribute to the immune defence in the human female lower genital tract. These parameters did not change during the menstrual cycle but hormonal contraceptive usage, especially DMPA, affected the quality of the epithelium. The use of DMPA and LNG was correlated with the accumulation of T cells within the epithelium. The effects of these changes on the risk of contracting infections are yet to be determined.

Retinal pigment epithelium (RPE) is a principal site of pathogenesis in age-related macular de-generation (AMD). AMD is a main source of vision loss even blindness in the elderly and there is no effective treatment right now. Our aim is to describe the relationship between the morphology of RPE cells and the age and genotype of the eyes. We use principal component analysis (PCA) or functional principal component method (FPCA), support vector machine (SVM), and random forest (RF) methods to analyze the morphological data of RPE cells in mouse eyes to classify their age and genotype. Our analyses show that amongst all morphometric measures of RPE cells, cell shape measurements (eccentricity and solidity) are good for classification. But combination of cell shape and size (perimeter) provide best classification.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

PCWE has been used widely in oriental traditional medicine in treating edema and diarrhea. Recent studies have shown that PCWE may also have anti-tumor and anti-inflammation acts. However, few studies have been conducted to reveal the mechanisms of these effects. In the present study, we tried to elucidate the mechanisms by investigating the effects on PCWE on the regulation of ion transport across the epithelial membranes of colon, which is also useful in explaining the anti-diarrhea and anti-edema effects. Alternation in membrane potential and short-circuit current (Isc) were examined using the Ussing chamber technique. Our results showed that PCWE decreased Isc upon application to the apical side. Amiloride inhibited this Isc induced by PCWE indicating that PCWE acted on amiloride-sensitive sodium channel of the epithelium. However, when PCWE was applied to the serosal side, the Isc was not changed, indicating a minimal influence of this substance on ATP-driving Na+/K+ counter transporters. Our data also showed that the Isc decreased by PCWE could be inhibited by bumetanide and chlorothiazide (Cl¡Â channel inhibitors). We therefore concluded that PCWE could both enhance sodium transport and stimulate the secretion of Cl¡Â in colon epithelium.

In the shark rectal gland (SRG), apical Cl[superscript]- secretion through CFTR channels is tightly coupled to a basolateral K[superscript]+ conductance. The identity of this K[superscript]+ conductive pathway is unknown. Studies were performed in the isolated perfused SRG with 16 K[superscript]+ channel inhibitors at their IC50 and with acidic perfusate. During maximal chloride secretion stimulated by forskolin and IBMX, secretion was inhibited >90% by barium chloride, a non-selective inhibitor of K[superscript]+ channels. Specific inhibitors of calcium sensitive, voltage sensitive, ATP sensitive, and inward rectifying K[superscript]+ channels had no effect on chloride secretion. The inhibitors quinidine, quinine, bupivicaine, anandamide, and low perfusate pH (6.0) abruptly and reversibly inhibited secretion by >90%, consistent with the presence of the Two-Pore-Domain (4TM 2P/KCNK/K2P) family of K+ channels. Degenerate primers were designed to regions of high amino acid homology in known mammal and teleost 4TM 2P K[superscript]+ channel subtypes: TWIK, THIK, TASK, TREK, and TRAAK. PCR with cDNA from several shark tissues identified a putative TASK-1 fragment (394 bp) in shark rectal gland, brain, gill, and kidney. 5and 3 RACE PCR was used to obtain the entire 3 sequence and partial 5 sequence of the shark gene. Genome walking was then used to obtain the remaining 5sequence, including 335 bp of untranslated region sequence upstream of the start codon. The full length clone (1282bp) had an open reading frame encoding a protein of 375 amino acids. This isoform was 80% identical at the amino acid level to the human TASK-1 protein (394 amino acids). Major structural features of the human protein were conserved in the shark ortholog, including the four transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2- and long COOH-termini, and an extended extracellular loop between M1 and P1. Shark and human TASK-1 full-length clones were expressed in Xenopus oocytes and studied with two electrode voltage clamp (TEVC) techniques. Both the shark and human TASK-1 channel showed identical current voltage relationships (outward rectifying) with a reversal potential near -90 mV compared to water injected controls. The responses to the inhibitor quinine, and the TASK-1 inhibitor bupivacaine, were identical in shark and human TASK-1. However, shark TASK-1 differed from the human ortholog in two critical responses: response to pH and the metal zinc. The pKa for shark TASK-1 was 7.75 vs. 7.37 for human TASK-1, values that are exceedingly close to the arterial pH for each species, suggesting that TASK-1 channels are regulated closely by the ambient pH. An antibody specific to shark TASK-1 was generated and expression of TASK-1 protein in the rectal gland was confirmed by confocal immuno-fluorescent microscopy which revealed localization to the basolateral membrane, with some apical staining. Shark rectal gland TASK-1 appears to be the major K[superscript]+ channel coupled to secretion in the SRG, is the oldest 4TM 2P family member identified to date, and is the first TASK-1 channel identified to play an essential role in chloride secreting epithelia.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains x, 148 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 136-148).

Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tumor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT ( BRCA1 185delAG Amino Terminal truncated protein), exhibit enhanced chemosensitivity and up-regulation of the OC-associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasion and metastasis. BRAT-expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting. Pro-MMP1 levels are also higher in conditioned media of BRAT-expressing cells and HOSE cell lines derived from BRAT mutation carriers. c-Jun is critical for BRAT-mediated MMP1 up-regulation, as siRNA knockdown diminishes MMP1 levels. Luciferase reporter constructs reveal that activator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT-mediated MMP1 expression, and basal activity is mediated in part by an AP1 site at (-72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Interestingly, BRAT-mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue-specific mutant BRCA1 functions may contribute to breast and ovarian tissue specificity of BRCA1 mutation-associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up-regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.

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